The invention relates to an interferometric apparatus for monitoring changes of the refractive index of fluid samples in capillary tubes, in particular to a refractive index based measurement system used for example in capillary liquid chromatography or capillary electrophoresis.
In modern analysis of chemical samples there exists a great demand for techniques involving only a small volume of the test sample. Capillary liquid chromatography and capillary electrophoresis belong to the most exciting and potentially useful microvolume separation techniques. Among the most important features of these new instrumental techniques are high peak resolution, short analysis time, and the minimal amounts of test sample required. The total column volume in these new techniques amount to as little as only a few microliters, and the required sample volumes are in the nanoliter or even in the picoliter range. Various instrumental aspects of these capillary-based, fluid-phase separation schemes contribute to the overall system performance. A major instrumental limitation of the performance of these techniques is the lack of highly sensitive microvolume detectors and the fact, that the so-called "off-column" detection, where the separated bands of the test sample are transferred to the detection cell, is associated with unwanted re-mixing effects.
As a solution to the problems associated with the "off-column" detection these capillary-based techniques employ so-called "on-column" detection methods, which do not distort the spatial profile of the eluting peaks. Among these "on-column" detection methods the monitoring of changes of the refractive index of the fluid test sample proves to be most promising. This technique is based on tile detection of tile phase shift of a coherent light beam passing through the fluid test sample, which occurs due to refractive index changes of the test sample with respect to the carrier buffer or the solvent contained within the capillary tube. The method makes use of the fact, that the phase shifts of the light beam transversing the fluid test sample flowing through the capillary tube are linearly related to the changes of the refractive index of the test sample.
For monitoring the changes of the refractive index of the test sample in capillary tubes a refractive index measurement system has been developed. The refractive index measuring system consists basically of an interferometer, having a source of coherent light, which is directed at the capillary tube, a photoelectric detector, and an evaluation electronics. The light beam, usually coming from a laser source, strikes the capillary tube; part of the light beam transverses the flowing path of the test sample and interacts with the test sample; whereas part of the light beam is reflected at the glass walls of the capillary. Thus, the light beam is split at the capillary tube, more specifically at its inner optical interface, into a probe beam and into a reference beam having a phase difference, which upon recombination of the two beams results in a generally asymmetric interference fringe pattern in the far field. The interference fringe pattern is measured by the photoelectric detector in the forward direction of the probe beam, and shifts laterally as the refractive index of the test sample flowing through the capillary tube changes.
Refractive index measuring systems of the kind described before are known from the prior art. The performance of these measuring systems was distinctly increased by arranging the capillary tube within material, which matches or almost matches the refractive index of the material the capillary tube is made from, as described in an article by A. E. Bruno, B. Krattiger, F. Maystre and H. M. Widmer, Anal.Chem., 1991, 63, page 2689-2697. In doing so, the boundary optical interfaces, which the laser beam encounters upon its propagation along the light path and striking the capillary tube, are reduced, the interference fringe pattern becomes more simple and easier to evaluate, and the signal/noise ratio is enhanced. In spite of the good results, which are achieved with the thus improved refractive index measuring systems known from the prior art, these devices still can be further improved. In case the capillary tube is not surrounded by a matching material, the resulting interference fringe pattern is rather complex and cannot be reliably evaluated and interpreted. Also, irregularities on the boundary surfaces of the capillary tube may modify the reference beam in an unexpected way.